The role of p53 as a tumor-suppressor protein is well known and is based on its ability to transcriptionally activate effector proteins that cause cell cycle arrest in G1 that allows time for repair of DNA damage. An alternate cellular choice is apoptosis to eliminate cells with damaged genomes. Loss of p53 activity by mutation is a major predisposing factor to transformation and cancer. The consequences of p53 and loss of function are appreciated in the cancer field but the functional significance of p53 in other diseases is not well studied. A major question in the p53 field continues to be how cell signaling decides between cell cycle arrest versus apoptosis. This question is a particularly important for possible p53 contributions towards programmed cell death in neurodegenerative processes and diseases are less recognized but are likely to be very influential in neuronal stress and injury. Our laboratory has been studying p53-mediated cell death in neuronal cell models (rat AF5 and rat PC12 cells) by reactive oxygen species (ROS) using hydrogen peroxide and nitric oxide. These studies are being conducted in both proliferating and differentiated cell states to test the hypothesis that p53 involvement in cell death will be different during propagating and differentiation states. We have collaborated with Dr. Freed at NIDA/NIH in Baltimore using his recently developed rat AF5 neuronal cell line to test this hypothesis. AF5 neural cells are centrally-derived neuronal cells that have been immortalized with a modified SV40 LT vector but still express wild type p53. We wanted to examine if these neuronal cells responded to oxidative stress-induced DNA damage stimuli with hydrogen peroxide and to signaling disruption with the kinase inhibitor, staurosporine. Results of the study recently published by McNeill-Blue et al., (2006) found that both agents cause apoptosis but by different mechanisms. We found that hydrogen peroxide produced hallmarks of apoptotic cell death from 24-48 hr at about 90% loss of viability. DNA fragmentation, caspase3/7 activation, cytochrome c release and Annexin V immunofluorescence and cell cycle arrest were well correlated with biochemical responses of p53 increase, activation by ser15 phosphorylation of p53 and luciferase p53 reporter responses and downstream dependent gene expression such as the apoptotic proteins, bax, Noxa and PUMA. While staurosporine effectively DNA fragmentation indicating apoptosis, it did so rapidly within 8-12 hr but without Annexin V externalization. Staurosporine increased p53 and ser15 phosphorylation and upregulated downstream p53 targets p21(waf1) and apoptotic proteins, bax, Noxa and PUMA. Interestingly, a large proportion of AF5 cells recovered, in contrast to hydrogen peroxide treatment, and were actively dividing by 24 hr. These findings demonstrate that AF5 neuronal cells undergo apoptosis in response to oxidative stress but differently in signal pathway disruption by STSP in a manner that would be useful in studies related to p53-dependent neuronal cell death and neurodegeneration. Continued studies by Dr. McNeill-Blue focus on the mechanism of p53 activation by ERK kinase in cell death pathways. In a second ongoing neuronal cell project, we hypothesize a role for p53 in differentiation using NGF-induced differentiation (neural growth factor) in rat PC12 cells. p53 transcriptional activity by reporter assay is increased differentiation at which time cells undergo growth arrest and neurite formation. A genome-wide p53-chromatin binding screen has identified multiple p53 genomic binding sites of the p53 protein. In part, p53 subserves differentiation by growth arrest but other genes have become activated. Validation studies have also identified p53 gene targets directly associated with differentiation. Both studies support a role for p53 in apoptosis and growth arrest. A fundamental question to be answered are the transcriptional mechanisms by which activated p53 supports growth control or apoptosis. Results of these studies will be published and presented at the Society of Neurobiology annual meetings.